SIMULTANEOUS ESTIMATION OF HYDROCHLOROTHIAZIDE, AMLODIPINE BESYLATE AND TELMISARTAN IN COMBINED TABLET DOSAGE FORM BY USING RP- HPLC METHODHTML Full Text
SIMULTANEOUS ESTIMATION OF HYDROCHLOROTHIAZIDE, AMLODIPINE BESYLATE AND TELMISARTAN IN COMBINED TABLET DOSAGE FORM BY USING RP- HPLC METHOD
Priyanka Kulkarni and Deepali Gangrade *
Vivekanand Education Society’s College of Pharmacy, Mumbai University, Chembur (E), Mumbai, Maharashtra, India.
ABSTRACT: A simple, selective, rapid, precise reverse phase high pressure liquid chromatographic method has been developed for the simultaneous estimation of Hydrochlorothiazide, Amlodipine besylate and Telmisartan in tablet dosage form as per ICH guidelines. The wavelength selected for the analysis was 239 nm for amlodipine besylate, telmisartan and hydrochlorothiazide. The solvents used were methanol and distilled water. The mobile phase 0.05M Potassium dihydrogen ortho-phosphate (pH-3.2), acetonitrile and methanol in the ratio 45:45:10 was used to carry out the separation. Column used was Supelco C18 (250 x 4.6 mm, 5μ). Flow rate was selected as 1ml/min. Retention time for Hydrochlorothizide (HCT), Amlodipine besylate (AML) and Telmisartan (TEL) was found to be 2.9min., 5.1min., 8.2min. The developed method was validated for accuracy, precision, linearity, robustness, ruggedness, specificity. The proposed method can be used for the estimation of these drugs in combined dosage forms.
RP-HPLC, Hydrochlorothiazide, Amlodipine besylate, Telmisartan, Validation
INTRODUCTION: The chemical name for Amlodipine besylate is 3-ethyl 5-methyl 4RS-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl - 1, 4-dihydropyridine - 3, 5-dicarboxylate benzene sulphonate. 1 Amlodipine besylate is the Calcium channel blocker. 2 It is used as an anti-hypertensive and in the treatment of angina. Amlodipine besylate is a white crystalline powder. It is slightly soluble in water and sparingly soluble in ethanol. It is official in BP, EP, IP, USP.1, 3, 4, 5
Methods available for the determinations of AML include UV spectroscopy 6, 7, 8, High performance liquid chromatography 9, 10, 11, High performance thin layer chromatography 12, 13, LC‐MS 14, LC ‐ MS/ MS 15, and stability indicating assay method.16 The chemical name for HCT, 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine - 7 - sulphonamide 1, 1-dioxide. It is a thiazide diuretic.17 It is also used in the treatment of hyper-tension.18
HCT is White to off-white crystalline powder.19 Soluble in dilute ammonia, or sodium hydroxide; also soluble in methanol, ethanol, acetone.20 It is official in Indian Pharmacopoeia, British Pharmacopoeia and United States Pharmacopoeia. 21, 17, 22 Methods available for determination of HCT includes spectrophotometry 23, liquid chromatography 24, 25, stability indicating assay 26 method and thin-layer chromatography 27, as alone or in combination with some other drugs.
Telmisartan is is chemically 4′-[(1,4′-dimethyl-2-propyl [2,6′-bi-1H-benzimidazol]-1′-yl) methyl] [1,1′-biphenyl]-2-carboxylic acid. 28 It is an Antihypertensive drug. 29 This is used for treatment of hypertension and diabetic nephropathy. 30, 31
It is official in Indian Pharmacopoeia (IP), British Pharmacopoeia (BP) and U.S. Pharmacopoeia (USP).32, 33, 34
Methods for determination of Telmisartan includes UV, 35, 36, 37 visible spectrophotometric, 38 Colorimetry 39, 40, liquid chromatography-tandem mass spectrometry 41, 42, 43, 44 and HPTLC.45, 46
MATERIALS AND METHODS:
Instrumentation: Shimadzu HPLC system with autosampler (model LC-2010-HT) having UV detector and column heater was used. Data collection and analysis were performed using LC Solution software. Other instruments used are mentioned in the Table 1.
TABLE 1: INSTRUMENTS USED FOR METHOD DEVELOPMENT
|Sr. No.||Name of the instrument||Company|
|2||Weighing balance||Premier weighing system|
|3||pH meter||DBK Digital pH meter|
Reagents & Chemicals: Pharmaceutically pure sample of AML, TEL and HCT were obtained as a gift samples from Smruti Organics; Glenmark Pharmaceuticals; IPCA Laboratories respectively. All solvents were of HPLC grade. A combination of AML (5 mg), TEL (40 mg) and HCT (12.5 mg) in tablet formulation was purchased from local market. (TELVAS-3D; Aristo Pharmaceuticals). All Reagents and chemicals used for method development are given in Table 2.
TABLE 2: REAGENTS AND CHEMICALS
|Name of Chemicals||Grade||Manufacturer|
|Acetonitrile||HPLC & Analytical||Pallav Chemicals|
|Methanol||HPLC & Analytical||Pallav Chemicals|
|Potassium Dihydrogen Orthophosphate||Analytical||LOBA Chemie|
|O-Phosphoric Acid||Analytical||Pallav Chemicals|
|Distilled Water||HPLC||MilliQ Water|
Chromatographic conditions: Column- Supelco C18 (250×4.6mm×5μ) was used for the separation. Mobile phase of buffer 0.05M sodium dihydrogen phosphate with pH 3.2, acetonitrile and methanol were mixed in the ratio 45:45:10, filtered through 0.45μ membrane filter, degassed and used for the separation. The flow rate was 1ml/min and inject volume was 20μL with detection at 239nm and analysis was performed. Chromatographic conditions are mentioned in the Table 3.
TABLE 3: CHROMATOGRAPHIC CONDITIONS
|Mobile phase||Buffer(pH 3.2): ACN: Methanol (45: 45: 10 v/v)|
|Column||Supelco C18, 250 x 4.6 mm, 5μ|
Preparation of Solutions:
- Preparation of Buffer Solution pH 3.2: Weigh accurately 6.8 gm of potassium dihydrogen phosphate and mixed it with 1000ml of water. Adjust the pH to 3.2 ± 0.05 with ortho- phosphoric acid and filter through 0.45μ nylon membrane and degas.
- Preparation of Diluent: Prepare a mixture of Water and Methanol in the ratio 50:50v/v respectively and mix.
- Preparation of Standard Solution: Weigh accurately about 12.5 mg of Hydrochlorothiazide, 40 mg of Telmisartan, 5mg of Amlodipine besylate API and transfer it into 100ml volumetric flask. Add about 70ml of diluent and sonicate to dissolve, then cool and make up to the mark with diluent and mix. Further dilute 1 ml of this solution into 10 ml volumetric flask and make up to the mark with diluent and mix. (Standard Solution 12.5μg/ml HCT, 40μg/ml TEL, 5μg/ml AML).
- Sample preparation: Weigh and transfer 10 tablets into 100ml volumetric flask. Add about 70ml of Diluent and sonicate with intermittent shaking. Allow it to cool at room temperature and make up to the mark with Diluent and mix. Further, dilute 1ml of that solution to 100ml with diluent and mix. Filter the sample solution through 0.45μ Nylon filter. Discard first 2ml of filtrate.
RESULTS AND DISCUSSION: Before validation of the developed method, analysis of blank solution (only mobile phase), standard solution (mixed standard) and sample solution (tablet mixture) was carried out. Following were the results obtained.
FIG.1: CHROMATOGRAM OF BLANK SOLUTION
Observation: It was found that the blank solution does not show any interference of characteristic
peak and therefore can be used for estimation of drugs.
FIG. 2: CHROMATOGRAM OF STANDARD SOLUTION 12.5μg/ml HCT, 40μg/ml TEL, 5μg/ml AML
FIG. 3: CHROMATOGRAM OF SAMPLE SOLUTION 12.5μg/ml HCT, 40μg/ml TEL, 5μg/ml AML
The method validation was done according to the ICH guidelines. The following validation characteristic parameters are accuracy, precision, linearity, robustness, ruggedness and specificity.
Accuracy: The accuracy of the method was determined by recovery experiments. The recovery studies were carried out six times and the percentage recovery and standard deviation of the percentage recovery were calculated and presented in Table 4, 5 and 6.
TABLE 4: SERIES OF ACCURACY SAMPLES PREPARED OF HCT FOR ASSAY METHOD
|Recovery Level %
|Amount of Sample Added (µg/ml)||Amount of
|Mean peak area||%
TABLE 5: SERIES OF ACCURACY SAMPLES PREPARED OF AML FOR ASSAY METHOD
|Recovery Level %
|Amount of Sample Added (µg/ml)||Amount of
|Mean peak area||% Recovery
TABLE 6: SERIES OF ACCURACY SAMPLES PREPARED OF TEL FOR ASSAY METHOD
|Recovery Level %||Amount of Sample Added (µg/ml)||Amount of
|Mean peak area||% Recovery
- % Recovery of HCT was found to be in the range of 100.47%- 101.35% and %RSD was found to be between 0.55%- 1.02%.
- % Recovery of AMLwas found to be in the range of 100.26%- 100.98% and %RSD was found to be between 0.75%- 1.09%.
- % Recovery of TEL was found to be in the range of 100.09%- 101.5% and %RSD was found to be between 0.34%- 0.45%.
- From the above observation it was concluded that the method is accurate for the simultaneous determination of HCT, AML & TEL in a tablet dosage form.
- Precision: The results of precision method were evaluated by carrying out six independent test samples of HTZ, AML and TLM. The percentage of RSD of six sample peak area values were calculated. The RSD values of intra-day and inter-day studies for HTZ, AML and TLM confirming good precision of the optimized method.
- a) Method Precision: The method precision of the method was established by carrying out the analysis of analyte (n=6) using the proposed method. The low value of relative standard deviation showed that the method was precise. The results obtained were presented in Table 7.
TABLE 7: SYSTEM PRECISION FOR ASSAY OF HCT, AML AND TEL
|Mean peak area (n = 6)||344646||341767||5520983|
- b) System precision: The system precision of the method was established by six replicate injections of the standard solution containing both the analytes of interest. The percentage RSD was calculated and presented in Table 8, 9.
Results for Intra-day Precision:
TABLE 8: METHOD PRECISION FOR ASSAY METHOD OF HCT, AML AND TEL
Results for Intra-day Precision:
TABLE 9: METHOD PRECISION FOR ASSAY METHOD OF HCT, AML AND TEL
DISCUSSION: The % RSD values indicate an acceptable level of precision of the analytical system for the assay of HCT, AML and TEL. The % RSD values on lower side indicate that there exists only minimum variation in the results and thus the system precision and method precision is validated.
Linearity: Linearity studies were performed at six different concentrations. The linearity data was calculated on individual drug basis and checked for the R2 value.
TABLE 10: LINEARITY DATA OF HCT FOR ASSAY METHOD
FIG. 4: LINEARITY CURVE FOR HCT
TABLE 11: LINEARITY DATA OF AML FOR ASSAY METHOD
FIG. 5: LINEARITY CURVE FOR AML
TABLE 12: LINEARITY DATA OF TEL FOR ASSAY METHOD
FIG. 6: LINEARITY CURVE FOR TEL
Discussion: Linearity was established for HCT within 6-36µg/ml, for AML within 2.5-15µg/ml and for TEL within 20-120µg/ml. Graphs of Concentration v/s Peak Area were plot. Regression equation for HCT, AML and TEL was obtained. Regression co-efficient for linearity of HCT is 0.9997, for AML 0.9994 and that for TEL is 0.9997.
Ruggedness: Ruggedness of the method was verified by analyzing six samples of the same concentration used for method precision as per proposed method by different analysts.
TABLE 13: EFFECT OF CHANGE IN ANALYST FOR HCT, AML AND TEL
|System Suitability Parameter||Observation||Limits|
|Drug||Analyst 1||Analyst 2|
|The % RSD of peak area response||HCT||1.24||1.28||
|Retention Time (min.)||HCT||2.983||2.953|
Discussion: The % RSD values for intermediate precision were found to be within the specified limit. This indicated that the method is rugged and can be used to analyze the formulations containing HCT, AML and TEL effectively in any given condition.
Robustness: Robustness of the method was evaluated by changing the flow rate by ± 10 %, by changing the wavelength by ± 5 nm and by changing the pH of buffer by ± 0.2 units.
Effect of Variation in flow rate of mobile phase by ± 10%:
TABLE 14: EFFECT OF VARIATION IN FLOW RATE OF MOBILE PHASE BY ± 10%
|Sr. No.||System Suitability Parameter||Observations||Limits|
|1.||The % RSD of peak area response||HCT||1.24||1.32||1.18||NMT 2%|
|2.||Theoretical plates||HCT||2503||2497||2598||NLT 2000|
|3.||Tailing Factor||HCT||1.235||1.352||1.206||NMT 2|
|4.||Retention Time (min.)||HCT||2.983||3.055||2.942|
Discussion: Small but deliberate variations were made in the method parameters and it was found that % RSD values for all the variations were in acceptable limits. This indicated that the method is
robust and that it can be used within small variations of flow rate, pH and wavelengths without having a major effect on the results of % assay values.
Effect of Change in wavelength of analysis by ± 5 nm:
TABLE 15: EFFECT OF CHANGE IN WAVELENGTH OF ANALYSIS BY ± 5 nm
|Sr. No.||System Suitability Parameter||Observations||
|1.||The % RSD of peak area response||HCT||1.24||1.35||1.12||NMT 2%
|2.||Theoretical plates||HCT||2503||2492||2540||NLT 2000|
|3.||Tailing Factor||HCT||1.235||1.224||1.315||NMT 2|
|4.||Retention Time (min.)||HCT||2.983||2.895||2.996|
Effect of Change in mobile phase pH by ± 0.2 units:
TABLE 16: EFFECT OF CHANGE IN MOBILE PHASE pH BY ± 0.2 UNITS
|Sr. No.||System Suitability Parameter||Observations||
|1.||The % RSD of peak area response||HCT||1.24||1.12||1.05||NMT 2%|
|2.||Theoretical plates||HCT||2503||2705||2698||NLT 2000|
|3.||Tailing Factor||HCT||1.235||1.325||1.250||NMT 2|
|4.||Retention Time (min.)||HCT||2.983||2.852||2.998|
- LOD (Detection limit): The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. For assay purpose limit of detection was not considered as the validation parameter as per ICH guideline
- LOQ (Quantitation limit): The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products. Therefore this is not considered as the validation parameter for assay purpose.
- Specificity: To ensure the absence of interference from blank in Hydrochlorothiazide, Amlodipine besylate and Telmisartan drug product. One blank solution of diluent was injected and Hydrochlorothiazide, Amlodipine besylate and Telmisartan were injected individually.
Results are shown in Fig. 1, 2 and 3.
Discussion: There was no interference from blank at the retention time of Hydrochlorothiazide, Amlodipine besylate and Telmisartan.
CONCLUSION: The method was developed and validated which concluded that the method is linear, simple, accurate, precise, rugged. Thus, the proposed method can be effectively applied for analysis of HCT, AML and TEL in bulk dosage forms as well as in combined tablet dosage form.
ACKNOWLEDGEMENT: The authors are thankful to Vivekanand Education Society’s College of Pharmacy, Chembur (E), Mumbai for providing all the necessary facilities required for the research work. The authors are also thankful to all the teaching and non-teaching staff and the colleagues for their constant support. Hearty thanks to our parents. Also authors are very thankful to IPCA Laboratories, Smruthi Organics and Glenmark Pharmaceuticals for providing gift samples.
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How to cite this article:
Kulkarni P and Gangrade D: Simultaneous estimation of hydrochlorothiazide, amlodipine besylate and telmisartan in combined tablet dosage form by using RP- HPLC method. Int J Pharm Sci Res 2017; 8(1): 268-76.doi: 10.13040/IJPSR.0975-8232.8(1).268-76.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Priyanka Kulkarni and Deepali Gangrade *
Vivekanand Education Society’s College of Pharmacy, Mumbai University, Chembur (E), Mumbai, Maharashtra, India
15 July, 2016
17 September, 2016
04 November, 2016
01 January, 2017